Background. The sympathetic nervous system (SNS) is responsible for hepatic stellate cells (HSCs) activation and the accumulation of collagen that occurs in hepatic fibrogenesis. Carvedilol has been widely used for the complication of hepatic cirrhosis in the clinic. Furthermore, it has powerful antioxidant properties. We assessed the potential antifibrotic effects of carvedilol and the underlying mechanisms that may further enhance its clinical benefits. Methods. Using a bile duct ligation rat model of hepatic fibrosis, we studied the effects of carvedilol on the fibrosis, collagen deposition, and oxidative stress based on histology, immunohistochemistry, western blot, and RT-PCR analyses. Results. Carvedilol attenuated liver fibrosis, as evidenced by reduced hydroxyproline content and the accumulation of collagen, downregulated TIMP-1 and TIMP-2, and upregulated MMP-13. MMP-2 was an exception, which was decreased after carvedilol treatment for 2 weeks and upregulated after carvedilol treatment for 4 weeks. Carvedilol reduced the activation of HSCs, decreased the induction of collagen, transforming growth factor-β1, and MDA content, and strengthened the SOD activity. The antifibrotic effects were augmented as dosages increased. Conclusions. The study indicates that carvedilol attenuated hepatic fibrosis in a dose-dependent manner. It can decrease collagen accumulation and HSCs activation by the amelioration of oxidative stress. Once activated, TGF-β1 binds to Smad proteins, which enhances the transcription of target genes, including procollagen I and procollagen III, and then leads to fibrosis development [Hepatic fibrosis, a reversible wound-healing response to a variety of stimuli, is characterized by the accumulation of extracellular matrix (ECM) mainly secreted by activated hepatic stellate cells (HSCs) . In normelopment .β-adrenergic blocker, carvedilol has been studied as an antioxidant [4) induced liver fibrosis, which can be explained by amelioration of oxidative stress [β-adrenoreceptor antagonist and antioxidant might be beneficial for hepatic fibrosis treatment.Oxidative stress also plays a major role in hepatic fibrogenesis, which is an important stimulus to HSCs activation . Reactivioxidant , 7. It hioxidant . Moreoveioxidant . Recentle stress . In concIn the present study, hepatic fibrosis model was established by bile duct ligation (BDL) in Sprague-Dawley (SD) rats. Carvedilol was administered in three dosages 48 hours after BDL for 2 weeks and 4 weeks, respectively. In addition to assessing the effects of carvedilol on liver fibrosis, we also investigated its potential mechanisms.Fifty adult male Wistar rats weighing 250–300 g were obtained from the Experimental Animal Center of Hebei Medical University (permission number 705188). The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the US guidelines . The experiment was performed in compliance with the national ethical guidelines for the care and use of laboratory animals (certificate number 911102).−1·d−1, CAR-L), medium-dose carvedilol treatment group , and high-dose carvedilol treatment group . Rat hepatic fibrosis model was established by applying BDL. Forty-eight hours after the operation, carvedilol was administered twice a day. Each group was also randomly divided into two groups, and their liver tissue specimens were separately taken 2 weeks and 4 weeks after BDL.The rats were randomly divided into five groups (10 rats per group): sham surgery control group, bile duct ligation (BDL) model group, low-dose carvedilol treatment group , aspartate aminotransferase (AST), total bilirubin (TBil), and albumin (Alb) were detected using a fully automatic biochemical analyzer. Liver specimens were fixed for 12–24 h in 4% phosphate-buffered paraformaldehyde  and then embedded in paraffin for light microscopy examination. Tissue sections (4 μm thick) were stained with hematoxylin and eosin (H&E) for morphological evaluation and Masson trichrome (MT) for assessing the degree of fibrosis.Blood was obtained from the left ventricular apex of rats and centrifuged at 3000μm thick liver tissues embedded in paraffin were deparaffinized and serially dehydrated in ethanol. Then, they were treated with 5% hydrogen peroxide for 10 min to inactivate the endogenous peroxidase. After 20 min of blocking with PBS containing 10% normal goat serum, the sections were incubated with primary antibodies. The primary antibodies included type I, III, and IV collagen  used as a marker for collagen expression; smooth muscle actin polyclonal antibody  used as a marker of activated HSC; TGF-β1  used as marker of cytokine. After treatment with HRP-conjugated goat anti-mouse IgG and 50 μL of streptavidin-peroxidase solutions for 30 min at RT, the sections were stained with DAB and counterstained with hematoxylin. The positive areas showed the color of brown yellow. Immunohistochemical analysis was performed with Image-Pro Plus.The 3 Collagen was detected by estimating the hydroxyproline content, an amino acid characteristic of collagen. Hepatic hydroxyproline was measured using a hydroxyproline detection kit  according to the manufacturer's instructions.β-actin 1 : 5,000; mouse monoclonal antibody; Sigma-Aldrich, St. Louis, MO), anti-matrix metalloproteinase-13 , antitissue inhibitors of metalloproteinase-1 , anti-MMP-2 (1 : 300), and anti-TIMP-2 (1 : 400). All the above were from Santa Cruz Biotechnology. Then, secondary antibody (Santa Cruz Biotechnology) was added, and the reaction bands of western blot were quantified by Quantity One software  and modified by the β-actin. The values (% of control) are given as means ± SD of 5 animals.Liver samples were homogenized with lysis buffer  and centrifuged at 20,000 ×g for 60 minutes at 4°C. The resultant supernatants were used as the total liver protein and subjected to western blotting. The protein concentrations were determined by Bradford's method. Proteins were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinyl difluoride membrane. Each blot was treated with the anti-b-actin antibody  by thiobarbituric acid reactive substances. The experiment was conducted according to the manufacturer's instructions . The conjugation was accompanied by an increase in absorbance at 532 nm. The cytosolic SOD activity was assayed using the SOD detection kit . The autooxidation rate of epinephrine, which was progressively inhibited by the increasing amounts of SOD in the homogenate, was monitored spectrophotometrically at 550 nm. The amount of enzyme that inhibited 50% of epinephrine autooxidation was defined as 1 U of SOD activity.P < 0.05. Results are expressed as means ± SD.Data were analyzed statistically by one-factor analysis of variance (ANOVA). Differences between experimental groups were analyzed using Student-Newman-Keuls (SNK) multiple-range test. The significant level was set at We found that the liver functions in hepatic fibrosis model groups were obviously damaged according to the expression of ALT, AST, TBil, and Alb. And the levels of ALT and AST in CAR-H groups were significantly lower than in model groups Tables  and 2. HType I, III, and IV collagen expressions were detected by immunohistochemistry staining. It was demonstrated that the expressions of type I, III, and IV collagens in carvedilol-treated groups were markedly reduced compared to BDL group, while no marked differences were found among the three carvedilol-treated groups Figures .μg·mg−1, 0.279 ± 0.031 μg·mg−1 versus 0.363 ± 0.027 μg·mg−1; P < 0.01, P < 0.01, resp.)  (P < 0.05).Hydroxyproline is unique to collagen and thus serves as a specific biochemical marker of collagen production. Two weeks after BDL, the hydroxyproline content in BDL group was markedly higher than that in the sham group. However, it was significantly decreased in CAR-M and CAR-H groups  . Four we, resp.) . And a sα-SMA expression in CAR-M and CAR-H groups was markedly reduced compared to that in BDL group   .We investigated the changes of HSCs in the present BDL rats to examine the effect of BDL-induced overactive sympathetic tone on the activation of HSCs, the key cell for the progressive accumulation of collagen in fibrosis disease. Immunohistochemistry staining for a-SMA, the marker of the activated HSCs, was mildly increased in the BDL group compared to that in sham group and was reduced by carvedilol treatment. After the 2-week carvedilol administration, the  Figures . After t Figures . And a sα-SMA immunohistochemical double staining. Among the 2-week groups, the apoptosis index of HSC was markedly higher in the CAR-M and CAR-H groups than in BDL group   . The results suggested that carvedilol may inhibit the activation of HSCs and promote apoptosis via the antisympathetic predominance pathway.HSCs apoptosis was determined by TUNEL and  Figures . Similar Figures . And a s versus 1.24 ± 0.09; P < 0.01), but it was significantly reduced in CAR-H group (1.33 ± 0.04). After carvedilol treatment for 4 weeks, the MMP-2 protein expression in CAR-M and CAR-H group was significantly higher than that in BDL group .The degradation of the matrix components markers MMP-2, TIMP-2, MMP-13, and TIMP-1 was used for the same rats in order to evaluate the accumulation and the degradation of matrix components based on the western blot and real-time Q-PCR . Two wee versus 1.1 ± 0.11; P < 0.05, P < 0.05, and P < 0.01, resp.). Similar results were shown in 4-week carvedilol groups. And a significant difference was found between the CAR-L and CAR-H groups (P < 0.05). The MMP-2 and MMP-13 mRNA expressions showed the same tendency as the protein expression above (data was not shown).After carvedilol treatment for 2 weeks, the MMP-13 protein expression in CAR-L, CAR-M, and CAR-H groups was significantly higher than that in BDL group . In the 4-week groups, TIMP-2 in CAR-L, CAR-M, and CAR-H groups was significantly reduced compared with that in the BDL group  . The results determined by RT-PCR were the same as the results above (the data was not shown). All these results suggest that carvedilol may modify the shift in the balance of the accumulation and degradation of matrix components in the BDL rats.TIMP-1 and TIMP-2, mainly produced by HSC, combine with active MMPs to inhibit their activity. Two weeks and 4 weeks after BDL, the protein expression of TIMP-1 and TIMP-2 in BDL group was increased compared with that in sham group. After BDL and carvedilol treatment for 2 weeks and 4 weeks, TIMP-1 protein expression in CAR-M and CAR-H groups was markedly reduced. In the 2-week groups, the protein expression of TIMP-2 in the CAR-H group was dramatically lower than that in the BDL group . But no statistical difference was found between the CAR-M and CAR-H groups. Four weeks after BDL, TGF-β1 expression in CAR-L, CAR-M, and CAR-H groups was significantly decreased compared with that in the BDL group  , while no statistical difference was found among the carvedilol-treated groups. Four weeks after BDL, there was no significant difference between the carvedilol-treated groups and the BDL group  showed that MDA contents in the CAR-L, CAR-M, and CAR-H groups were significantly lower than those in the BDL group , while no marked differences were found among the three carvedilol-treated groups. Similarly, four weeks after BDL, SOD activities in the CAR-L, CAR-M, and CAR-H groups were higher than those in the BDL group   .Carvedilol is a nonselective beta-blocker with potent antioxidant and free radical scavenging properties, which is used in the treatment of portal hypertension commonly associated with chronic liver diseases. Considering these powerful antioxidant properties of carvedilol, we predicted that carvedilol may exert an antifibrotic effect on chronic liver injury. This means carvedilol has additional clinical benefits. So, in the present study, we investigated the efficacy of carvedilol on BDL-induced hepatic fibrosis and the potential mechanisms involved. Three dosages of carvedilol treatment for 2 weeks and 4 weeks were used, which was beneficial for observing the curative effect on different stages of fibrosis.β is the mostly potent fibrogenic cytokine, which has three major isoforms . TGF-β1 is the principal isoform implicated in liver fibrosis. The activated TGF-β1 combines with Smad proteins via its cognate receptors, which enhances the transcription of target genes, including collagens I and III [β1 expression. Therefore, we inferred that the inhibition of TGF-β1 by carvedilol was one of the pathways for decreasing collagen expression.In the study, H&E and Masson staining confirmed the established hepatic fibrosis model. The liver inflammatory and fibrous formations were markedly alleviated by carvedilol treatment. Hepatic fibrosis is characterized by abnormal accumulation of ECM, which is changed not only in quality but also in quantity. In normal liver, the low-density basement membrane-like matrix in the space of Disse is mainly composed of collagens IV and VI. However, the fibrillar collagens including collagens I and III and fibronectin are markedly increased in hepatic fibrosis . This in and III , 12. Theα-SMA by several stimuli such as cytokines and inflammatory mediators. Activated HSCs migrate to and proliferate in sites of liver injury, synthesize ECM components, and upregulate the expression levels of α-SMA and collagen matrices [α-SMA is a marker of HSC activation and proliferation. The results of α-SMA expression showed that carvedilol administration inhibited HSC activation. Furthermore, α-SMA and TUNEL double staining demonstrated that carvedilol increased the apoptosis of activated HSC. Regulating the biologic behavior of HSC may be one of the reasons for carvedilol to prevent the progression of hepatic fibrosis.HSCs are typically found in the space of Disse in a quiescent state . HSCs mamatrices . Hence, Hepatic fibrosis occurs because of the synthesis and excessive deposition of ECM in the space of Disse along with insufficient ECM degradation. The equilibrium between MMPs and TIMPs mainly determines the homeostasis of ECM. MMPs are the main enzymes responsible for ECM degradation and TIMPs are their specific inhibitors . In the Bile duct ligation is an established method for investigating cholestasis and hepatic fibrosis, which leads to changes in the equilibrium between antioxidant and prooxidant activities . The depβ1 expression, increased HSCs apoptosis, and increased ratio of MMPs/TIMPs, as well as its antioxidant property. Consequently, carvedilol treatment as early as possible in the maximum dosage that patients are able to bear may be beneficial for preventing hepatic fibrosis progression.In summary, carvedilol plays a preventive role in a dose-dependent fashion in the progression of hepatic fibrosis induced by BDL, which is associated with decreased TGF-


Blood glucose control in the intensive care unit (ICU) has the potential to save lives. However, maintaining blood glucose concentrations within a chosen target range is difficult in clinical practice and holds risk of potentially harmful hypoglycemia. Clinically validated computer algorithms to guide insulin dosing by nurses have been advocated for better and safer blood glucose control.n = 777) or blood glucose control by trained nurses  during ICU stay, according to the local target range (80–110 mg/dL or 90–145 mg/dL). The primary outcome measure was the quality of blood glucose control, assessed by the glycemic penalty index (GPI), a measure that penalizes hypoglycemic and hyperglycemic deviations from the chosen target range. Incidence of severe hypoglycemia (<40 mg/dL) was the main safety outcome measure. New infections in ICU, duration of hospital stay, landmark 90-day mortality and quality of life were clinical safety outcome measures.We conducted an international, multicenter, randomized controlled trial involving 1550 adult, medical and surgical critically ill patients, requiring blood glucose control. Patients were randomly assigned to algorithm-guided blood glucose control . Mean blood glucose was 111 mg/dL (SD 15) in LOCIC-C versus 119 mg/dL (SD 21) in Nurse-C, whereas the median time-in-target range was 67.0% (IQR 52.1–80.1) in LOGIC-C versus 47.1% (IQR 28.1–65.0) in the Nurse-C group (both P < 0.001). The fraction of patients with severe hypoglycemia did not differ between LOGIC-C (0.9%) and Nurse-C (1.2%) (P = 0.6). The clinical safety outcomes did not differ between groups. The sampling interval was 2.3 h (SD 0.5) in the LOGIC-C group versus 3.0 h (SD 0.8) in the Nurse-C group (P < 0.001).The median GPI was lower in the LOGIC-C (10.8 IQR 6.2–16.1) than in the Nurse-C group (17.1 IQR 10.6–26.2)  contains supplementary material, which is available to authorized users. Elevated blood glucose levels are very common in critically ill patients, independent of pre-existing diabetes mellitus. This hyperglycemia has been associated with an increased risk of morbidity and mortality . Tight bExcess mortality in patients undergoing BGC has been attributed to the increased incidence of hypoglycemia and unnecessary blood glucose variability , 14. InaThe LOGIC-Insulin algorithm, developed and based on the Leuven guideline and practice, had already been clinically validated in a single-center randomized trial, showing improved and safer BGC (below 110 mg/dL), compared to that performed by nurses who were highly experienced in BGC . HoweverWe conducted a pragmatic, parallel-group, observer-blinded, randomized controlled trial, involving medical and surgical patients admitted to the ICUs of three hospitals: two tertiary referral academic centers  and one non-academic, teaching hospital . All ICUs involved have a closed organization, staffed by full-time intensivists, and represent, respectively, 98, 50, and 36 ICU beds.The study protocol and informed consent documents were approved by the Belgian Federal Agency for Medicines and Health Products (80 M0563) and the institutional review boards of each participating center. The trial was registered on ClinicalTrials.gov (NCT02056353) on 4 February 2014. No design changes occurred during the trial. Whenever possible, informed consent was asked for from the decision-competent patient before study inclusion. In the case of emergency ICU admissions, deferred proxy consent from the legal representative was obtained. The data and safety monitoring board (DSMB) reviewed the data twice according to the charter and advised continuation according to the initial protocol.Patients were recruited from 24 February 2014 to 17 December 2014. All patients admitted to the ICU with an expected stay of at least 2 days and already receiving or potentially needing insulin for blood glucose control were screened for eligibility  or the LOGIC-Insulin-guided (LOGIC-C) group by a central computerized system on a 1:1 ratio with permuted blocks of 10 per stratum. Block size was unknown to all collaborators. Randomization was stratified according to type of admission (post-cardiac surgery or other) and study center. Randomization of cardiac surgery patients was stopped after reaching 60% of the total study population across all centers. Outcome assessors, but not clinical staff, were blinded to treatment allocation.Patients received blood glucose control according to the assigned group: Nurse-C or LOGIC-C. An intravenous infusion of insulin was started on admission in both groups to aim for glycemia in the target range, which in the Leuven and Hasselt ICUs was glycemia 80–110 mg/dL, and in the Amsterdam ICU was glycemia 90–145 mg/dL. Blood glucose targets were identical for both diabetic and non-diabetic patients. In all centers and in both randomization groups insulin treatment was initiated when glycemia exceeded the upper limit of the target range.BGC was discontinued in both groups when the patients started oral intake of carbohydrates, when the arterial and/or central line was removed, when the patient was discharged to the general ward or to an external ICU, when palliative care was initiated, or when recurrent severe hypoglycemic episodes (glycemia <40 mg/dL) or refractory hyperglycemia (>180 mg/dL and insulin at the aforementioned maximum rate) were observed. Blood glucose concentrations were measured in undiluted arterial blood by an on-site blood gas analyzer, which differed between study sites. Insulin at a concentration of 50 IU in 50 mL of saline was infused through a central venous catheter, preferably via a dedicated lumen, using a syringe pump.BGC in the Nurse-C group was based on the existing local documented guidelines. All bedside nurses had undergone training in blood glucose management during their schooling as an ICU nurse in the different units. No specific training in BGC was given to nurses performing BGC in the Nurse-C group for the purpose of this study. Hence there was a range of experience in dealing with insulin in the context of BGC. The bedside nurse decided on the timing of blood glucose sampling and the adjustment of the insulin infusion rate according to the best clinical practice.The LOGIC-insulin algorithm guided BGC in the LOGIC-C group. The LOGIC-Insulin software is an open loop system that advises the nurse on the dose of insulin administration, or on dextrose in the case of hypoglycemia, and on the timing of the subsequent blood glucose measurement. It also entails visual warnings about sampling time, hypoglycemia and potential user entry errors, such as nutrition. All LOGIC-Insulin advices have to be confirmed by the bedside nurse and, if appropriate, can be overruled. The heart of the LOGIC-Insulin algorithm itself is a robust, biphasic, and adaptive patient model. The first phase consists of two main parameters: patient profile on the one hand and admission variables, such as diabetes mellitus and the severity of illness, on the other hand. The second phase comprises five variables: patient profile, blood glucose, insulin dose sequence, the administration of steroids, and nutrition.Further, the algorithm includes both feedback and predictive mechanisms allowing estimation of the effect of future disturbance factors , 26. TheThe LOGIC-Insulin algorithm was designed and configured in the pre-study phase to deal with varying blood glucose target ranges. Since there was a 1:2 to 1:3 nurse-patient ratio in the participating hospitals, nurses may have had to treat patients in the two different groups: Nurse-C and LOGIC-C.The primary outcome measure of the LOGIC-2 trial was quality of blood glucose control, assessed by the glycemic penalty index (GPI) per patient, during the intervention period, censored at 14 days. This index gives a penalty to all glucose values falling in the hypoglycemic and hyperglycemic zones with a higher penalty value for larger deviations from normoglycemia . The aveThe other outcome measures of BGC were in line with the recent consensus recommendations on reporting of glycemia in critically ill patients . The mosOther secondary endpoints were the incidence of mild hypoglycemia (<70 mg/dL), mean arterial blood glucose concentration, hyperglycemic index (area under the curve in the hyperglycemic zone), time-in-target range, time-to-reach-target range, maximal blood glucose difference (marker of blood glucose variability) and the interval between blood glucose measurements (marker of workload), all reported as per-patient metrics with the exception of the incidence of mild hypoglycemia, which is presented both as the incidence per patient and the incidence per proportion of measurements. In the intervention group protocol compliance (patients in whom the LOGIC-insulin software was not followed for a period of at least 8 h) and overrules (recommendations that were not followed by the bedside nurse) of the software were assessed. A distinction was made between minor (absolute insulin dose difference of > 0.1 IU/h and <1 IU/h) and major (≥1 IU/h) overrules.Clinical safety endpoints were the incidence of new infections in the ICU , ventilator days per patient (censored at 14 days), length of stay in the ICU and in the hospital, mortality in ICU and in hospital, and the landmark 90-day mortality. Quality of life was assessed using the EuroQol 5D-3 L questionnaire at ICU admission, ICU discharge and 90 days post-randomization. Sepsis was diagnosed, using the American College of Chest Physicians-Society of Critical Care Medicine criteria .The study was conceived as a superiority trial for improving the quality of blood glucose control, measured by GPI and the time-in-target range, and it was also powered to detect differences in the incidence of mild hypoglycemia (<70 mg/dL) as a safety outcome variable. The sample size calculations were preregistered at the Belgian Federal Agency for Medicines and Health Products. Better performance in BGC in the Nurse-C group was anticipated, as nurses would feel they were to be watched (Hawthorne effect). On the basis of a 5% confidence level (α error) and a 90% statistical power (β error 10%) the study required 458 patients in each arm to detect a decrease in GPI from mean 21 to 18 (sigma 14) in a two-sided test. An increase in the time-in-target range from 45% to 50% with a sigma 29% would require 707 patients per arm. For a decrease in mild hypoglycemia from 27% to 20% (ARR 7%) the study required 769 patients in each arm. To take into account withdrawals, the study was set up for 1550 patients (775 patients in each group).A subgroup analysis per study center and blood glucose target was preplanned. The following subgroup analyses for specific patient populations were also planned: cardiac surgery, medical, sepsis on admission, infection on admission, and known diabetes mellitus.Variables were summarized as frequencies and percentages, mean and standard deviation (SD) or median and interquartile range (IQR), as appropriate. Confidence intervals were computed based on the bootstrap percentile method for the primary and secondary endpoints .2) (Fisher exact) test, Student t test, or nonparametric (Wilcoxon rank sum) test as appropriate. For all endpoints, differences were considered statistically significant whenever the two-sided P value was <0.05, without correction for multiple testing. For the statistical analyses, JMP Pro 11  and Matlab  were used.All analyses were done on an intention-to-treat basis. Data were compared using the chi-square  gave consent and were randomized to either Nurse-C or LOGIC-C  (Table P < 0.001). Mean blood glucose levels and the hyperglycemic index were also lower in the LOGIC-C group . Moreover, blood glucose variability was decreased in the LOGIC-C group (P < 0.001). The proportion of patients experiencing at least one episode of hypoglycemia did not differ between treatment groups . However, the proportion of blood glucose readings <70 mg/dL and <60 mg/dL was smaller in the LOGIC-C group (both P = 0.02). None of the subjects in either randomized group experienced recurrent severe hypoglycemic episodes or refractory hyperglycemia that warranted withdrawal from the study. Workload was higher in the LOGIC-C group, as reflected in a 23% shorter sampling interval (P < 0.001).The GPI, the primary outcome measure, was 6.3 points lower in the LOGIC-C group than in the Nurse-C group (1) Table . Time-inThe effect of the LOGIC-Insulin algorithm on BGC was more pronounced in Hasselt and Amsterdam Fig. . While tThe quality of BGC was improved by the algorithm in the predefined subgroups of cardiac surgery . The median daily amount of carbohydrates was 42 g (IQR 31–68) in the LOGIC-C and 42 g (IQR 30–70) in the Nurse-C group (P = 0.88). The number of LOGIC-C patients who received steroids (24.1%) was comparable to the Nurse-C group (21.7%) (P = 0.28). Also the proportion of patients on mixed-bag parenteral nutrition did not differ between the LOGIC-C (7.3%) and the Nurse-C (10.0%) groups (P = 0.07). The number of days patients received inotropics/vasopressors  and antibiotics  did not differ between treatment groups (both P > 0.39).Patients in the LOGIC-C group received a larger daily insulin dose (median 32 IU/day IQR 18–48 versus median 27 IU/day IQR 14–43 in the Nurse-C group) (P < 0.001).In 80/777 patients (10.3%) in the LOGIC-C group, the software had not been used for more than 8 h. In total 240 minor and 147 major overrules occurred in the LOGIC-C group, representing 1.15% and 0.70% of blood glucose measurements, respectively. Of the major overrules, 14 (9.5%) were justified, and of these only 2 overrules were made to avoid hypoglycemia, representing 2/777 LOGIC-C patients (0.3%). In Amsterdam the proportion of overrules (minor 19 (1.71%), major 20 (1.80%) for insulin dosing advice given in 1114 instances) was higher than in Leuven (minor 155 (1.30%), major 89 (0.75%) for insulin dosing advice given in 11926 instances) and Hasselt (minor 66 (0.84%), major 38 (0.48%) for insulin dosing advice given in 7892 instances) (P = 0.034). In patients with an infection on admission, the incidence of new infections was 23.04% in the LOGIC-C, compared with 31.96% in the Nurse-C group (P = 0.042). For all other predefined subgroups  the incidence of new infections was comparable between treatment groups.The clinical outcomes did not differ between the treatment groups  than in the narrow target range of 80–110 mg/dL (delta GPI 5.7) was therefore not anticipated. One might have expected that BGC is easier for nurses when the target is broader. This indicates that nurses, in the context of a broader target range, tend to be more lenient towards the upper target limit. The higher incidence of extended hyperglycemia above 180 mg/dL reflects this, in spite of the consensus in all guidelines on BGC in the ICU that excessive hyperglycemia should be avoided. Nevertheless, the higher incidence of overrules in Amsterdam, possible practical differences in the insulin administration, and greater statistical uncertainty due to the smaller patient numbers may have contributed as well. Most likely, BGC will be even looser outside study settings. In contrast, use of the narrower target range results in greater incidence of mild hypoglycemia. Algorithm-guided BGC did not increase the incidence of potentially harmful, severe hypoglycemia, which should always be strictly avoided. A recent randomized controlled trial used computerized insulin-dosing algorithms in the intensive BGC group, but did not show a clinical difference with more conservative, nurse-directed BGC .Algorithm-guided BGC increased the workload, similar to other algorithms . Ten insThe improvement in BGC provided by the software was comparable in all patient populations, such as cardiac surgery, pre-existing diabetes, sepsis, and medical admissions. However, the clinical effects of improved BGC should be the ultimate determinants to evaluate medical interventions. Although the LOGIC-2 trial was not set up to test differences in clinical outcomes, it was monitored. As expected, no clinical differences were seen in the overall patient population. Patients with diabetes mellitus did not benefit more from algorithm-guided BGC, as was seen in a recent trial of BGC in patients after coronary artery bypass graft surgery . HoweverIn patients with infectious problems on admission, improved BGC resulted in lowering of the incidence of new infections. This may be explained by the fact that patients with infectious problems on admission are more severely ill, have greater risk of more severe hyperglycemia, which is more difficult to control, and have greater risk of new infections.The LOGIC-2 trial has its limitations though. Blood glucose dynamics in the ICU are determined by patient characteristics, by the BGC protocol, and by the experience of the bedside nurses in executing the BGC. Therefore, the differences in the quality of BGC in the Nurse-C group between the centers are most likely multifactorial. The variability in the “baseline” quality of BGC may have affected the performance using the LOGIC-Insulin software. It can also not be excluded that nursing practice in performing BGC changed over time during the study, under the influence of the algorithm. However, participating in a study on BGC had most likely improved the performance of BGC by the nurses over time due to a training effect. The LOGIC-Insulin software was now only tested when using glucose readings from arterial blood measured in accurate blood gas analyzers. To gain further applicability the software should be able to cope with blood glucose measurements from point-of-care, handheld blood glucose meters. Other computerized protocols however, have already used these point-of-care glucometers showing a safe and efficient level of blood glucose control , 24. EffIn a randomized controlled trial of a mixed critically ill patient population, the use of the LOGIC-Insulin blood glucose control algorithm, compared with blood glucose control by expert nurses, improved the quality of blood glucose control without increasing hypoglycemia. This is demonstrated by an important improvement in all domains of glycemic control: a decrease in GPI, an increase in the time-in-target range, and a decrease in glucose variability. Moreover, the LOGIC-Insulin algorithm was shown to be efficient and safe when used in three different ICUs with local variations in clinical practice, with different blood glucose target ranges, and with different levels of experience in blood glucose control. The slight increase in workload generated by the LOGIC-insulin algorithm should be considered an inevitable trade-off when providing safe and efficient blood glucose control.Additional file 1:Tables showing predefined subgroup analyses. (DOCX 50 kb)Additional file 2:Study protocol: Competent Authority submission. (PDF 8786 kb)


P = 0.598). No statistically significant differences were observed in intra-abdominal abscesses, stump leakage, superficial wound infections, post-operative abdominal pain, overall adverse events, or the duration of the operations and hospital stays between the groups . Adverse risk factors associated with the use of absorbable clips in LA included body mass index ≥ 27.5 kg/m2, diabetes, American Society of Anesthesiologists degree ≥ III, gangrenous appendicitis, severe inflammation of the appendix base, appendix perforation, and the absence of peritoneal drainage. The results indicate that the Lapro-Clip is a safe and effective device for closing the appendicular stump in LA in select patients with appendicitis.A randomized control trial was performed to evaluate the effectiveness and safety of absorbable polymeric clips for appendicular stump closure in laparoscopic appendectomy (LA). Patients were randomly enrolled into an experimental group  or control group . We identified 1,100 patients who underwent LA between April 1, 2012 and February 3, 2015. Overall, 99 patients  developed a complication following LA (47 [8.5%] in the L-C group and 52 [9.5%] in the H-C group ( Laparoscopic appendectomy (LA) is frequently performed to treat appendicitis . It is aA total of 1,100 patients were recruited to our study between April 2012 and February 2015. Of these patients, 550 received Lapro-Clips to ligate the appendicular base (L-C group), while the other 550 patients received Hem-o-lok Clips to ligate the stump (H-C group). There were no significant differences in patient age, gender, body mass index (BMI), or American Society of Anesthesiologists (ASA) degree between the two groups (Table p = 0.228). Overall adverse events included all complications, re-interventions, and other adverse events . No significant differences in other adverse events were identified between the L-C and H-C groups . Finally, no patients died as a consequence of either surgical approach.There were 122 adverse events observed in the L-C group and 139 in the H-C group (P = 0.326). There were no significant differences in the category of appendicitis, degree of inflammation of the base of the appendix, perforation (not in the base), peritoneal drainage, or histology (P > 0.05) between the two groups. All of these data are presented in Table In the L-C group, the average diameter of the appendix base was 8.52 ± 2.03 mm (range 3.5–11.0). In contrast, the average diameter in the H-C group was 8.27 ± 2.13 mm (range 3–10) (P = 0.598). An intra-abdominal abscess (IAA) developed in 29 patients (5.3%) in the L-C group and 33 patients (6.0%) in the H-C group (P = 0.179). No statistically significant differences were observed in stump leakage, superficial wound infection, or post-operative abdominal pain between the two groups   developed a complication after LA (47 [8.5%] in the L-C group and 52 [9.5%] in the H-C group (tients 5.% in the P = 0.425). A total of 11 patients (2.0%) in the L-C group underwent additional surgery because of IAA or stump leakage, while 13 patients (2.4%) underwent additional surgery in the H-C group (P = 0.680). No statistically significant differences were observed in percutaneous and/or transrectal drainage, prolonged administration of intravenous antibiotics, or readmission between the two groups (P > 0.05).Re-interventions were required in 52 patients (9.5%) in L-C group compared to 60 patients (10.9%) in H-C group (P = 0.679). The mean length of hospital stay was 3.56 ± 2.11 days in the L-C group and 3.49 ± 2.33 days in the H-C group (P = 0.531). There were no significant differences in the duration of surgery or the length of hospital stay between the two groups.The average duration of surgery (from the time the incision was made to the time of skin closure) was 41.4 ± 19.8 min in the L-C group and 40.6 ± 20.3 min in the H-C group (P < 0.05). Gender did not influence the prognosis of patients with appendicitis following surgery (P = 0.980). Peritoneal drainage could effectively reduce the risk of adverse events. According to the statistical analyses, one clip was safe for appendicular stump closure  , and peritoneal drainage impacted the incidence of adverse events –180 days . This means there will be no foreign body remaining in the enterocoelia following LA. Metal clips, non-absorbable polymeric clips, and endostaplers may interfere with imaging methods such as X-ray, computed tomography, and magnetic resonance imaging. The absorbable clip potentially provides long-term benefits to patients following surgery because it avoids the impact of foreign bodies. The Lapro-Clip could not be used in patients with a large appendix base (> 11 mm) or severe inflammation in appendicular base. However, in the present study, it can be used in most patient populations. The considerations related to Lapro-Clips use are also applicable to other devices such as endoloops and metal or non-absorbable polymeric clips. However, we found that the Lapro-Clip could also be safely applied in some patients with an inflamed appendix base if the appendix base was only slightly or moderately inflamed. Recent studies have indicated that endoloops can be used in patients with an appendix base ≤ 15 mm, titanium clips in patients with a base of ≤ 20 mm, and Hem-o-lok Clips in patients with a base of ≤ 10 mm , 18, 24.In 14.5% of patients, two clips were used at the appendix stump while in 85.5% of patients, surgeons used only one clip. It was advisable to transfer the appendix immediately into a retrieval bag in order to avoid contamination. We did not find significant differences in adverse events between the single-clip and double-clip groups, which indicated that the use of one clip was sufficiently safe in LA.Typical complications associated with appendicitis include IAA and stump leakage. Peritoneal drainage reduces the rate of IAA significantly , 26, 27.The use of clips instead of staplers has resulted in reduced costs. In our study, the price of one absorbable clip was approximately 274Y ($43.50) and the price of one Hem-o-lok clip was 148.5Y ($23.60). The mean cost of LA was 9362Y  in the L-C group and 9297Y  in the H-C group, respectively. The costs of LA in both groups (including the readmission fees) were lower than those reported by other studies , 31.2, diabetes status, ASA degree ≥ III, gangrenous appendicitis, severe inflammation of the base of the appendix, perforation of the appendix, and the absence of peritoneal drainage, which indicates surgeons should use endostaplers instead of clips in patients with these risk factors to reduce the rate of complications and adverse events.In this study, we evaluated risk factors for both absorbable and non-absorbable polymeric clips in stump closure in LA. Based on the data, we conclude that the risk factors included BMI ≥ 27.5 kg/mThe limitation of this study was that patients with different category of appendicitis, especially those with IAA and/or gangrene in appendix were enrolled, which may result in higher adverse events and potential bias. The way to make the conclusion more reliable may enroll patients with purulent appendicitis but without gangrenous appendicitis. Besides, although the result indicated that the outcomes are similar in 1 and 2 absorbable clips groups, it may conduct potential bias. Thus, more strict study design is necessary.The Lapro-Clip is a safe and effective device for closing the appendicular base in LA. The absorbable clip shows a comparable complication rate and adverse event rate to the Hem-o-lok Clip. Thus, the Lapro-Clip is a suitable tool for LA that does not result in residual foreign bodies and does not increase the economic burden of patients. There are some limitations of the Lapro-Clip in patients with a severely inflamed appendix and/or a large base. Surgeons should evaluate the safety of using the Lapro-Clip according to the risk factor model.The study was a randomized control trial. Prior to beginning the study, we planned to randomly recruit 1,100 patients with appendicitis to the patient cohort based on statistical considerations  who could meet the inclusion criteria were informed of the approach the surgeons would use to close the appendicular stump and were also told that their data  would be documented in the study. The inclusion and exclusion criteria are shown in Figure All surgeons participating in this study could perform appendicular closure with Lapro-Clips or Hem-o-lok Clips proficiently. Participating physicians were instructed to close the appendicular stump with one or two clips based on their own clinical judgment and to submit information related to the parameters and outcomes of the treatment to the database.The absorbable clip is made of absorbable polymeric materials including poly hydroxy acid and poly glucose ester . The clips are absorbed by the body within 90 days  and 180 days . Lapro-Clip devices are intended for use in laparoscopic cholecystectomy and other procedures in which clips are indicated Figure .In this study, the product was used within the indication. Furthermore, no additional examinations or interventions were performed on the patients. Therefore, this study was not considered to constitute an additional risk for enrolled patients. Ethical approval was obtained from the appropriate ethics committees before patients were enrolled in the study. Written informed consent was obtained by the investigator at each center from all patients prior to enrollment.LA and pre-operative or concomitant treatments were performed according to the standards. LA was performed using the three-port approach. The Lapro-Clip was used according to the manufacturer's instructions. Before the study began, the hospital was informed about the study procedures. Surgeons resected the mesoappendix and the artery of the appendix using an ultrasonic knife without any other management. This knife was also used to remove the appendix. The application of one clip to the stump was recommended and estimated to be sufficient for closure. The surgeon decided whether or not to use an additional clip Figure , 2C. TheThe primary outcome variables were the following: 1) Post-operative complications including IAA, superficial wound infection, appendicular stump leakage, and post-operative abdominal pain, which was defined as abdominal complaints after surgery requiring prolonged clinical observation or additional biochemical or radiological tests; 2) Re-interventions including percutaneous and/or transrectal drainage, re-operation (laparoscopy/laparotomy), and prolonged use of intravenous antibiotics (> 3–5 days); 3) Duration of the operation (the time from skin incision to skin closure), duration of hospital stay, and readmission .As follow-up, a telephone interview was conducted 30 days after surgery. Adverse events, re-admissions to the hospital, and medical treatments related to the operation that were required were recorded.T-tests were performed for continuous data. We considered p values < 0.05 to be statistically significant.All the data obtained during this study were tabulated and subjected to analysis using the standard methods of statistical analysis. Statistical analyses were performed using SPSS, version 19.0. Chi-squared tests were performed to evaluate proportional differences between the two groups.


Through microbial community analyses, we determined that an operational taxonomic unit (OTU) for Bacteroides spp. was dominant and was positively correlated with increased MCC productivities. Our data also suggested that the microbiome may have been shaped for improved MCC production by the high broth-recycle rates. Comparable abiotic studies suggest that further increases in the broth-recycle rates could improve the overall mass transfer coefficient and its corresponding MCC production flux by almost 30 times beyond the maximum that we achieved. With improved in-line extraction, the chain-elongation biotechnology production platform offers new opportunities for resource recovery and sustainable production of liquid fuels and chemicals.To convert wastes into sustainable liquid fuels and chemicals, new resource recovery technologies are required. Chain elongation is a carboxylate-platform bioprocess that converts short-chain carboxylates (SCCs)  into medium-chain carboxylates (MCCs)  with hydrogen gas as a side product. Ethanol or another electron donor  is required. Competitive MCC productivities, yields (product vs. substrate fed), and specificities  were only achieved previously from an organic waste material when exogenous ethanol had been added. Here, we converted a real organic waste, which inherently contains ethanol, into MCCs with n-caprylate as the target product. We used wine lees, which consisted primarily of settled yeast cells and ethanol from wine fermentation, and produced MCCs with a reactor microbiome. We operated the bioreactor at a pH of 5.2 and with continuous in-line extraction and achieved a MCC productivity of 3.9 g COD/L-d at an organic loading rate of 5.8 g COD/L-d, resulting in a promising MCC yield of 67% and specificities of 36% for each n-caprylate and n-caproate (72% for both). Compared to all other studies that used complex organic substrates, we achieved the highest n-caprylate-to-ncaproate product ratio of 1.0 (COD basis), because we used increased broth-recycle rates through the forward membrane contactor, which improved in-line extraction rates. Increased recycle rates also allowed us to achieve the highest reported MCC production flux per membrane surface area thus far (20.1 g COD/m The yietanoate) . Therefoiculture ; as fragiculture ; and as iculture  and aviaiculture . Longer- density . As a re density .n-Caproate has been produced with reactor microbiomes from ethanol (n-caproate productivities (n-caprylate has only been produced when ethanol was the electron donor (n-caprylate production during sustained n-caproate production (n-caprylate at high productivities (19.4 g COD/L-d) by using a high ratio of ethanol to acetate (15 g COD/g COD)  conoduction ; (3) higoduction ; (4) addoduction ; (5) andD/g COD) .n-caprylate and n-caproate without exogenous ethanol. We utilized a reactor microbiome as the inoculum for this study, which had already been shaped in a bioreactor that was fed a complex organic substrate . In addition, we tested whether increased bioreactor broth-recycle rates through the forward membrane contactor of the in-line extraction system could lead to improved undissociated MCCA extraction rates, and consequently, improved MCC production. Finally, we monitored the reactor microbiome during a phase of the operating period that included the increase in the broth-recycle rates.Here, our objective was to provide the first demonstration of the conversion of a real organic waste  into We obtained ∼75 L of wine lees during one collection from Sheldrake Point Winery . The winemaker described the lees as “gross settling yeast lees from the initial fermentation,” which represents ∼4% of the volumetric production of their wine . We measured an ethanol concentration of 180.5 g COD/L   system. During Phase II, we fed 1 L of substrate every other day, resulting in an HRT of 9 days and a total OLR of 6 g COD/L-d. We also increased the broth-recycle rate from 9 L/d in Period 2 to 323 L/d in Period 4, and we then decreased this recycle rate to 228 L/d in Period 5 ; semi-continuous substrate addition ; and continuous substrate addition  . For temperature control, we recirculated heated water from a water bath (37 ± 1°C)  through the glass jacket surrounding the bioreactor. A pH probe  was mounted through the bioreactor head plate. Automated pH control of the bioreactor broth was maintained at pH 5.2 with a controller  and a corresponding acid addition pump . When the controller provided hydrochloric acid (0.5 M) to the bioreactor, it also initiated automated headspace gas recirculation  through the bottom of the bioreactor to enhance mixing and prevent excessive acid addition. Several glass spheres (1.8-cm diameter) were placed in the cone at the bottom of the bioreactor  proceeded first through an in-line screening and filtration system and then continued through an in-line pertraction system that is described below. During the initial 58 days only (Periods 1–2), we augmented this broth recycle with an additional flow (140 L/d) that did not pass through the in-line screen, filtration, and pertraction systems to increase mixing inside the bioreactor. The upflow hydraulic velocities in the bioreactor were similar  , according to We used an in-line pertraction system with forward and backward membrane contactors . To prevent entrainment , we provided enough backpressure on the shell side of each membrane contactor to maintain slight trans-membrane pressure gradients (∼7 kPa). Undissociated MCCAs were then transferred across the backward membrane contactor and accumulated as dissociated MCCs in the alkaline extraction solution. The alkaline extraction solution was recycled at a flow rate of 7 L/d (Cole-Parmer 7553-30) from a stirred container through the shell side of the backward membrane contactor . This alkaline extraction solution initially had a volume of 2 L and was buffered with 0.3 M sodium borate. It was maintained at pH 9 with automated addition of 5 M sodium hydroxide using a controller  and a corresponding base pump .To extract undissociated MCCAs across the hydrophobic membrane, we used a hydrophobic mineral oil solvent. We added 30 g/L TOPO  to this hydrophobic solvent to encourage preferential extraction of the more hydrophobic undissociated carboxylic acids . The hydrophobic solvent was circulated continuously at a flow rate of 6 L/d (Cole-Parmer 7553-30) through the interior (lumen) of the membrane fibers in each membrane contactor (via standard methods (Liquid samples (1.5 mL) were collected from the bioreactor broth recycle and the alkaline extraction solution daily or every other day. We collected two samples from the bioreactor broth-recycle line between the 5-μm filter and the forward membrane contactor, and one sample was collected from the well-mixed reservoir of the alkaline extraction solution. We determined the concentrations of carboxylates and ethanol in these samples with separate GC systems . The con methods .g for 4 min, and the supernatants were discarded. The pelleted biomass samples were stored at -80°C until further processing. Genomic DNA was extracted using the PowerSoil DNA isolation kit . Modifications to the protocol included utilization of custom bead tubes containing a mixture of 300 mg of 0.1-mm diameter and 100 mg of 0.5-mm diameter silica/zirconia beads . Next, we calculated the washout rates of carboxylates by multiplying the average bioreactor broth concentrations of carboxylates with the eﬄuent flow rates. Subsequently, we calculated the total production rates of carboxylates (g COD/d) by summing the extraction and washout rates. To calculate the volumetric production rates (productivities), we divided the production rates by the working volume of the bioreactor (g COD/L-d). To calculate the extraction and production fluxes (g COD/m2-d), we divided the rates (g COD/d) by the membrane surface area  of the forward membrane contactor. Finally, we reported productivities as average values for each operating period. With exceptions noted, uncertainty was represented by 95% confidence intervals, as described previously  are: ethanol, 96; acetate, 64; propionate, 112; eviously .2-d). We predicted the extraction rates of undissociated MCCAs using a correlation, which we developed, between overall mass transfer coefficients  and bioreactor broth-recycle superficial velocities . We also used reported data, including: forward membrane contactor surface areas ; and concentrations of undissociated MCCAs in the bioreactor broth . Next, we calculated the washout rates for each study by: (1) multiplying the bioreactor volume (L) by the bioreactor broth concentration of MCCs (g COD/L); and then (2) dividing this multiplicative product (g COD) by the HRT (d). The total production rates, productivities, and fluxes were calculated as described above.In a separate comparative section, we used data from previous studies to predict and compare MCC production fluxes through the forward membrane contactor , we divided the broth-recycle rate by the cross sectional area of the forward membrane contactor shell (Across = 1.56 × 10-3 m2). When we varied the recycle superficial velocity (Phase II), we calculated the overall mass transfer coefficient (k) by dividing the extraction rate  by the multiplicative product of: (1) the membrane surface area ; and (2) the concentration gradient of undissociated MCCAs . This gradient represents the difference between the concentrations of undissociated MCCAs in the bioreactor broth and the alkaline extraction solution. Due to its high pH, the alkaline extraction solution contained negligible concentrations of undissociated MCCAs, which allowed us to estimate the concentration gradient as the bioreactor broth concentration of undissociated MCCAs.To calculate the recycle superficial velocity . For the first time, promising MCC production was performed from a real organic waste without the addition of exogenous ethanol. During Period 8 of Phase III, we achieved a maximum MCC productivity of 6.9 g COD/L-d, but the bioreactor was overloaded at a very high OLR of 51.9 g COD/L-d . The corresponding n-caprylate-to-n-caproate product ratio was 0.6 (COD basis) . Since the OLR was 5.8 g COD/L-d, we achieved an MCC yield of 67% with n-caprylate and n-caproate specificities of 36% (each) during Period 4  in a single-stage bioprocess, the authors achieved MCC productivities up to 1.8 g COD/L-d . This probserved . TherefoFigure 2A; Supplementary Table n-caprylic acid in the bioreactor broth , which are known to inhibit microbial activity (n-caprylic acid (C8) concentrations increased to 0.06–0.11 g COD/L (0.17–0.31 mM) during both the batch phase (Phase I) and in the phase of continuous substrate addition (Phase III)  concentration was 0.11 g COD/L (6% of its maximum solubility), which was only surpassed by our other published study (0.22 g COD/L)  concentrations remained ∼10-fold lower than 2.7 g COD/L (10.5 mM) that had been observed to inhibit reactor microbiomes  , overall substrate conversion , and MCC yield . To support chain elongation, in-line extraction can prevent undissociated MCCAs from accumulating to inhibitory levels in the bioreactor broth, which can otherwise hinder chain elongation pathways and thermodynamic feasibility.During the study by activity . Importag COD/L)  wherein robiomes . In addi–400 mM) . Therefon-caprylic acids, we observed that almost all ethanol was converted . This resulted in relatively low residual ethanol concentrations of 0.3 g COD/L (3.1 mM) in the broth . After, we decreased the HRT from 9.5 days (Period 5) to 1.2 days (Period 6) under similar operating conditions, the conversion efficiency of solids declined to 25% . Clearly, the short HRTs (∼1 day) that we used during Phase III (Periods 6–9) led to insufficient residence times for hydrolysis of solids. For future work with solids-rich substrates , we recommend a much longer retention time to promote hydrolysis. For example, for the yeast fermentation beer conversion in anaerobic fermenters, the HRT was ∼15 days  within this biotic study to evaluate its impact on the undissociated MCCA extraction rates. Because we simultaneously extracted a mixture of undissociated MCCAs, we based the extraction rates, concentrations, and overall mass transfer coefficients on the lumped MCCA products of n-caprylic acid and n-caproic acid in g COD. We knew from previous work that increasing the recycle rates for the hydrophobic solvent or the alkaline extraction solution through the membrane contactors did not improve the undissociated MCCA extraction rates. By varying the broth-recycle rates, we confirmed a fundamental relationship that governs undissociated MCCA extraction rates: the broth-recycle superficial velocity (u) through the forward membrane contactor was directly proportional to the overall mass transfer coefficient (k) . The recycle superficial velocity (u) is the speed at which bioreactor broth flows past the membrane surface, and it is known that higher speeds increase turbulence and decrease resistance to MCCA transfer across the membrane  (Atransfer) and undissociated MCCA concentration gradient (ΔCMCCA) , this relationship suggests that higher broth-recycle rates would improve undissociated MCCA extraction rates. Previously, an abiotic n-caproic acid extraction study employed a larger forward membrane contactor (Atransfer = 8.1 m2 and Across = 6.09 × 10-3 m2) and also established a linear correlation between the superficial velocity and the overall mass transfer coefficient   is directly proportional to the overall mass transfer coefficient (k) for Membrana Liqui-Cel X50 membrane contactors, regardless of their size.During Phase II , we varied the broth-recycle rate through the forward membrane contactor (raction) . Therefofficient . We obseMCCA) and fluxes by an order of magnitude . The maximum recycle superficial velocity (u) that we applied was 207 m/d (k = 50 mm/d) . From several previous abiotic membrane-based extraction experiments, much higher superficial velocities were used, which led to improved overall mass transfer coefficients (-2 m/s) was employed in a previous study, which resulted in a mass transfer coefficient of ∼4,500 mm/d , we anticipate that an overall mass transfer coefficient of more than 1,400 mm/d could be achieved at a superficial velocity of 6,000 m/d . Because of the improved MCC productivity (and improved substrate consumption), the average concentration of ethanol decreased with increased recycle rates, especially during Periods 4 and 5 when the recycle superficial velocities were above 100 m/d . Thus, improved in-line extraction rates would allow systems to direct substrate conversion toward increasingly hydrophobic, energy-dense, and valuable products.After we increased the broth-recycle rates during Periods 2–5, we also observed increases in the MCC productivities and Figure 6). To provide a common basis for comparison, we calculated the MCC production flux (g COD/m2-d) for each study. Here, we achieved the highest MCC flux compared to the other studies during Period 8 (up to 20.1 g COD/m2-d), albeit under overloaded conditions with poor MCC yields . During an earlier period , we supplied a lower organic loading flux (19.9 g COD/m2-d), which still resulted in an MCC production flux (13.3 g COD/m2-d) that was higher than all previous reports . Moreover, the n-caprylate-to-n-caproate product ratio from Period 4 (1.0 g COD/g COD) surpassed all other published studies that had used a complex organic substrate. We then proved that the correlation  could be used to predict the observed MCC production fluxes from several studies . Using only reported flow rates, equipment sizes, and bioreactor broth concentrations, the predicted MCC fluxes were reasonably well correlated to the observed MCC fluxes with an R2 of 0.74 . Higher n-caprylate-to-n-caproate product ratios  in n-caprylic acid is more hydrophobic and easier to extract than n-caproic acid. The predictive model is based on a lumped MCCA concentration parameter that was developed with comparable extraction rates of n-caprylic acid and n-caproic acid from the bioreactor broth . Therefore, this model should be further improved in future studies when n-caprylate comprises the majority of the MCC production.We compared the MCC production rates from several reactor microbiome studies that employed in-line extraction  for our bioreactor setup. In addition, increased broth-recycle rates directed more substrate toward elongated carboxylate products. Future optimization studies should now be commenced to ascertain the maximum improvements that can be made with a fixed membrane surface area.Figure 7). For all 12 samples, we observed 2526 OTUs from high-quality sequence reads. For the bioreactor samples, 36 OTUs exceeded 1% relative abundance in at least one sample. For the inoculum and substrate samples, an additional six and seven unique OTUs, respectively, exceeded 1% relative abundance. The total of these 49 OTUs accounted for 86.9 to 96.6% of the total high-quality sequence reads for each sample. The number of OTUs within the community and their relative proportions  did not vary considerably between microbiome samples from the operating period . In addition, we included one sample each from the inoculum and the substrate . Even though the inoculum was derived from a similarly operated bioreactor that was fed ethanol-rich yeast fermentation beer, we observed that the most dominant OTUs in the wine lees-converting bioreactor were relatively scarce in the inoculum . In addition, our substrate (wine lees) contained a unique microbial community compared to the bioreactor broth samples . As an open-culture biotechnology platform, we had not attempted to modify or suppress the microbiome in this substrate. Rather, ecological selection for MCC production was achieved by operating the bioreactor under controlled environmental parameters .The 49 OTUs were hierarchically ranked based on both the average relative abundance and the abundance profile throughout Phases I and II. Consequently, grouped OTUs in the heat map had similar shifts in abundances for the inoculum, the substrate, and throughout the operating time of the bioreactor (Lactobacillus spp. were predominant (21.9 to 50.3%) . Next, the relative abundances of these OTUs declined after in-line extraction was initiated at the beginning of Phase II. Concurrently, we observed increases in relative abundances of the OTUs for Ruminococcus spp. and other unknown species of the order Clostridiales and family Clostridiaceae . The broth-recycle rate through the forward membrane contactor was increased from a very low level in Period 2 to increasingly higher levels during Periods 3 and 4, and then back down to an intermediate level in Period 5 . When recycle rates had been increased to their maximum value during Period 4, an OTU for Bacteroides spp. surged in relative abundance on Day 74 . Period 4 also corresponded with the highest MCC productivity and maximum n-caprylate-to-n-caproate product ratio observed in this study. Accordingly, we determined that the MCC productivity was positively correlated with the relative abundance of Bacteroides spp. (p = 0.01), as well as with two less abundant OTUs for Oscillospira spp. (p = 2.5 × 10-5) and Clostridium spp. (p = 0.02) . In previous studies with similar inocula, we had also observed positive correlations between the MCC productivity and the relative abundance of the OTUs for Bacteroides spp. and Oscillospira spp. when the electron donors were lactate and ethanol, respectively (During the batch phase (Phase I), OTUs for ectively ,b.Figure 8). We observed a chronological order from early samples (lighter circles) to late samples (darker circles). One discrepancy in this chronological order was observed for Periods 4 and 5  . The sample from Day 84 was actually more similar to the sample from Day 64 than for the sample from Day 74. We had increased the broth-recycle rate from Period 2 through Period 4 (Day 74), and then we decreased the recycle rate during Period 5 again (Day 84) . Therefore, we suggest that the larger distance between the sample from Period 4 (Day 74) and earlier samples was due to the increased broth-recycle rate. The most dissimilar sample from our study (Day 74) represented the sample from the bioreactor with the highest broth-recycle rate, MCC productivity, and n-caprylate-to-n-caproate ratio , identifying that the microbiome had likely been shaped for its function. However, a future study would be needed to substantiate this finding.We also analyzed the dissimilarity of the OTU composition between pairs of samples by using the weighted UniFrac algorithm (beta diversity) , resultin-caprylate and n-caproate (72% for both). Compared to other studies that used complex organic substrates, we also achieved the highest n-caprylate-to-n-caproate product ratio of 1.0 (based on COD). Microbial community analyses indicated that OTUs for Bacteroides spp., Oscillospira spp., and Clostridium spp. were positively correlated with increased MCC productivities. At the end of the study, when we increased the substrate loading, undissociated n-caprylic acid concentrations prevented further increases in the MCC productivity due to product inhibition. We used data from biotic and abiotic studies to suggest that increased broth-recycle superficial velocities could have further increased the MCC productivities to almost 30 times the current maximum level with a fixed membrane surface area. Our data also shows that increasing the superficial velocities will change the microbiome.In a reactor microbiome with continuous in-line extraction, we provide the first demonstration of MCC production from a real organic waste (wine lees) without exogenous ethanol addition at a promising productivity of 7 g COD/L-d and a specificity of 36% for each -2 m/s). These new experiments will require careful analysis  and system optimization . We also encourage the development of a detailed TEA to guide technology deployment. Specifically, economic guidance is required to: (1) optimize between increased capital and operating costs; (2) select appropriate (waste) substrates; and (3) develop marketable MCC products. With continued development, we believe that chain elongation with in-line extraction can provide a new route for resource recovery and the sustainable production of liquid fuels and chemicals from wastes.To advance the chain-elongation biotechnology platform, we encourage new in-line pertraction experiments at high bioreactor broth-recycle rates  and the EBI database .16S rRNA sequence data and metadata are available on QIITA (LA proposed the study, and provided guidance to all co-authors. LK and JX: constructed the bioreactor system; designed and conducted the experiment; collected samples; and determined analyte concentrations. LK analyzed bioreactor system data and conducted calculations. MN performed all methods to characterize the microbial community, including corresponding data analysis and figure preparation. LK and JX prepared other figures. LA and LK wrote the manuscript, with revisions from JX and MN.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lutzomyia longipalpis (Lutz & Neiva 1912), a vector of visceral leishmaniasis. Max has a powerful vasodilatory effect and is a candidate vaccine that has been tested in experimental leishmaniasis. Nyssomyia neivai (Pinto 1926) is a vector of the pathogen responsible for American tegumentary leishmaniasis (ATL) in Brazil. Maxadilan (Max) is a salivary component in the sandfly Ny. neivai and for antibodies against Max in ATL patients. We searched for Max expression in Ny. neivai and analysed by polymerase chain reaction, DNA sequencing, and blotting assays. The results were compared with data obtained from Lu. longipalpis samples. We quantified antibodies against Max in serum samples from 41 patients with ATL  and 63 controls from the endemic northeastern region of São Paulo state, using enzyme-linked immunosorbent assay. cDNA and protein were extracted from the cephalic segment, including salivary glands, of Ny. neivai (GenBank EF601123.1). Compared to controls, patients with ATL presented higher levels of antibodies against Max (p = 0.004); 24.4% of the patients with ATL and 3.2% of the controls presented anti-Max levels above the cutoff index (p = 0.014). The anti-Max levels were not associated with the specific clinical form of ATL, leishmanin skin test response, absence or presence of amastigotes in histopathologic exam, results of indirect immunofluorescence testing for leishmaniasis, or duration of cutaneous form disease. Recognition of a Max-simile peptide by specific antibodies confirmed expression of a Max sequence in Ny. neivai bites in this ATL endemic region. High serum anti-Max levels did not protect patients against ATL, but confirmed previous natural exposure to  Leishmania (Kinetoplastida: Trypanosomatidae). The parasite is transmitted by the bite of phlebotomine vectors (Diptera: Psychodidae: Phlebotominae) that inoculate promastigote forms of Leishmania, along with saliva, into the host’s skin while acquiring a blood meal . This species has been implicated in the transmission of Leishmania (Leishmania) infantum chagasi (Cunha & Chagas 1937), the main causative agent of visceral leishmaniasis (VL) in Brazil  is a 7-kDa peptide present in the salivary glands of the sandfly Nyssomyia intermedia (Lutz & Neiva 1912) (= Lu. intermedia), a vector of the pathogen responsible for American tegumentary leishmaniasis (ATL) in Brazil, is highly divergent - its shows only 34% sequence identity with Max from Lu. longipalpis and is less abundant , a vector of cutaneous leishmaniasis (Leishmania infection (The Max homologue in Leishmania (Viannia) braziliensis (Viana 1911) and Leishmania (Leishmania) amazonensis (Lainson & Shaw 1972) are the species associated with the cutaneous and mucocutaneous forms of ATL (Autochthonous cases of ATL have been reported in the northeastern region of the state of São Paulo. Ny. intermedia and Ny. neivai (Pinto 1926) (= syn. Lu. intermedia) are the main vectors of Leishmania (V.) braziliensis in the state of São Paulo. However, only Ny. neivai has been identified in systematic collections in the municipalities of that region  was diluted in phosphate buffered saline (PBS) to a final volume of 2,000 mL and distributed into 500-mL aliquots. On the first day, 500 mL of complete Freund’s adjuvant  was mixed with an aliquot of Max, and a rabbit was injected with the mixture in each hip. Nine days later, another dose was injected in the foreleg and shoulder of the rabbit. On day 20, a third dose was injected at four points subcutaneously. Rabbit serum was tested by counterimmunoelectrophoresis on a 1% agarose gel before and after immunisation (positive pole) against Max (negative pole). Runs were performed using sodium barbital buffer in an electric current of 30 mA for 60 min, and then agarose gel was stained with Coomassie Blue .Sandflies and RNA-DNA-protein extraction - Laboratory-colonised Lu. longipalpis were provided by Alda Hawk, PhD, Fiocruz-Belo Horizonte, state of Minas Gerais, Brazil; Ny. neivai specimens were wild caught and identified by Claudio Casanova, SUCEN-Mogi Guaçu, state of São Paulo, Brazil. The cephalic segments, including the salivary glands, of Lu. longipalpis and Ny. neivai specimens were used for extraction by the Trizol method. In this report, the sandfly species names are presented in accordance with Galati’s classification system  and proteins extracted from Lu. longipalpis (0.02 mg/uL) and from Ny. neivai (0.01 mg/mL) were run in a 12.5% acrylamide gel using a Mini Protean II ; the acrylamide gel was stained by the silver method.Immunoblotting - Max and protein extracts submitted to electrophoresis were transferred to a 0.2-mm nitrocellulose membrane  . Then, each strip was incubated with a serum sample (1:20) for 2 h. After washing with thiosulphate-citrate-bile salts-sucrose (TCBS), a protein G-horseradish peroxidase (HRP) conjugate was incubated with the strips for 2 h, which was followed by colour revelation with an HRP-conjugated substrate .Comparison of the Ny. neivai - Max DNA sequence with that of Lu. longipalpis using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The Max DNA fragment was amplified with specific primers : sense-GCCATAGATGACTGCCAGAAGC and antisense-TTCCAGGTAGTTGGGAGGTATCC. PCR was performed in a thermocycler  with a final volume of 25 mL: 2.5 mL of 10 × buffer, 5 mL of 2 mM dNTP, 10.3 mL of distilled water, 1 mL 100 pmol of each primer, and 20 mL of Taq polymerase. A total of 35 cycles were run as follows: one cycle at 93ºC for 30 sec, one cycle at 94ºC for 3 min and 30 sec, one cycle at 52ºC for 1 min, one cycle at 72ºC for 1 min, and a final cycle at 72ºC for 10 min. Amplification of a 106-bp product was confirmed in a 10% acrylamide gel. HhaI and RsaI enzyme restriction was performed. HhaI acts at the GCG^C restriction site, generating 30-bp and 80-bp sequences. RsaI acts in GT^AC, resulting in two 50-bp sequences.Reverse transcription-PCR (RT-PCR) - RNA (12 µL) extracted from both sandflies species was added to 0.7 mL of RNase inhibitor, 4 mL of 5× RT buffer, 1 mL of 10 mM dNTP, 0.6 mL of RT , and 1.7 mL of distilled water at 42ºC for 90 min, followed by storage at -20ºC. cDNA was used in the PCR assay described above.Sequencing of PCR products - PCR was performed with the sense primer-CGTGTTTGCCTTCAGTAAGTTCT to amplify a long fragment and for sequencing. A 246-bp fragment was sequenced following the manufacturer’s recommendations .Study population - Patients were identified at the University Hospital, Ribeirão Preto Medical School, University of São Paulo, Brazil, which is the main reference institution for diagnosis and treatment of ATL in the northeastern region of the state of São Paulo. A diagnosis of ATL was confirmed with at least two of the following criteria: clinical-epidemiological diagnosis compatible with ATL; positive leishmanin skin test  defined as induration ≥ 5 mm 48-72 h after injection; histopathology of a skin or mucosa sample indicative of ATL, with or without visualisation of the amastigote form; indirect immunofluorescence (IIF) test ; and PCR result positive for the L. Viannia subgenus  standardised with Max peptide - Briefly, Max diluted in carbonate-bicarbonate buffer (125 mg/mL) was used to coat a microplate  at 4ºC for 48 h. After washing with PBS-Tween 20 (T20), 50 mL of each serum sample diluted in PBS-TM is Phosphate-buffered saline (PBS) plus Tween 20 plus Milk, plus 5% fat free milk (1:50) was incubated at 37ºC for 2 h. After washing, peroxidase-conjugated anti-human IgG  was incubated with plates at 37ºC for 2 h, and 100 µL of chromogenic substrate  was incubated with plates at room temperature in the dark for 15 min, followed by the addition of 50 mL of stop solution (1N H2SO4). Colour development was observed in an ELISA reader  at 492 nm. Anti-Max values were expressed as index values: the optical density (OD) of each sample was divided by the cutoff .Statistical analysis - Patients’ data are summarised the t-test was used to compare the results of two groups. Categorical variables were compared by Chi-square (χ2) test. Fisher’s exact test was used when data were sparse. Significance was set at p < 0.05; a two-tailed comparison was employed. The statistical analyses were performed using GraphPad Prism 6 software.Ethics - This study was approved by the Human Ethics Committee (#5.886/2002) of the University Hospital of the Ribeirão Preto Medical School, University of São Paulo, Brazil, in accordance with the ethical standards of the Helsinki Declaration  of the World Medical Association. All patients included in the study signed to indicate their informed consent prior to participation.Immunoblotting indicated Max-simile peptide in the Ny. neivai protein extract - Similar molecular weight fractions, including a 7-kDa peptide, were present in both the Ny. neivai and Lu. longipalpis protein extracts (data not shown). Immunoblotting with rabbit serum following immunisation against Max and Max peptide confirmed the immunisation specificity observed in the counterimmunoelectrophoresis. Subsequently, Max-immunised rabbit serum incubated with Ny. neivai protein extract confirmed a protein fraction with a molecular weight similar to that of Max . The patients’ median age was 45 years (minimum of three-years-old and maximum of 65-years-old); 80.5% of the patients were male. The median duration of the disease in the patients with ATL was six months (minimum of one month and maximum of 300 months). The time for disease evolution was greater in patients with the mucocutaneous form than in patients with the cutaneous form of ATL  (< 0.001) .Anti-Max antibodies levels  were higher in the ATL patients than in the controls (p = 0.0043) . Based oOver the last few decades, the number of ATL cases has been increasing worldwide. In the Americas, the number of ATL cases has grown markedly, and Brazil has contributed the majority of the cases . The staLu. longipalpis has not been identified in the northeastern region of the state of São Paulo, and to our knowledge, autochthonous cases of VL have not been reported in this region  and that this fragment shares 66.5% identity with Max from Lu. longipalpis.s region . In the ão Paulo  with a Mão Paulo , has notão Paulo . TherefoNy. intermedia transcriptome, its Max homologue is very divergent (34% identity) and the protein is present in low amounts compared to Max in Lu. longipalpis. As for Lu. ayacuchensis, another vector of cutaneous leishmaniasis in the New World, its transcriptome does not show Max gene expression, but large amounts of other vasodilator molecule genes such as adenosine and AMP are expressed  braziliensis and Leishmania (L.) amazonensis when the saliva is inoculated together with the parasite  (= Lu. flaviscutellata) and Psychodopygus complexus  (= Lu. complexa), vectors of Leishmania (L.) amazonensis and Leishmania (V.) braziliensis in the Brazilian Amazonian Region, respectively, exerts an inhibitory effect on parasite infection, resulting in small lesions and low levels of skin parasitism  they were bitten less frequently by sandflies than can be explained by epidemiological and environmental variables such as rural or urban occupation; (ii) they presented strong cellular immune responses ad weak humoral response against Max, resulting in against ATL; or (iii) they are genetically protected against ATL. It would be interesting to have data on the LST response in these individuals. A prospective study with blind evaluators could solve the limitations of our study.Ny. neivai saliva components in ATL endemic regions. Some authors have also shown that vector density and the presence of antibodies against vector saliva are correlated (Ny. neivai saliva components may be alternative serological markers to monitor ATL epidemiology in endemic regions. High titres of antibodies against Max in patients with ATL indicate previous exposure to vector bites, and, in our region, they represent previous exposure to the Max-simile peptide of Ny. neivai.Our data may encourage monitoring of human exposure to Max and to rrelated . AntibodNy. neivai contribute to the development of new diagnostic approaches, a better understanding of ATL, knowledge of vectors in this endemic region. The examination of anti-Max antibodies in patients with ATL and controls confirms previous exposure to Ny. neivai sandfly bites and suggests natural, chronic exposure to vectors. Therefore, antibodies against Max did not protect the host against ATL and could even enhance disease in our region. Further studies are essential to assess polymorphisms and the abundance of Max in Ny. neivai salivary glands. A description of the sialotranscriptome would also be of value.Studies on the genetic and protein expression of Max-simile in


Salix matsudana cv. Zhuliu) lignin. DESs types, mole ratio of ChCl to HBD, extraction temperature, and time on the fractionated DES-lignin yield demonstrated that the optimal DES-lignin yield  with high purity of 94.5% can be reached at a ChCl-to-Lac molar ratio of 1:10, extraction temperature of 120 °C, and time of 12 h. Fourier transform infrared spectroscopy (FT-IR) , 13C-NMR, and 31P-NMR showed that willow lignin extracted by ChCl-Lac was mainly composed of syringyl and guaiacyl units. Serendipitously, a majority of the glucan in willow was preserved after ChCl-Lac treatment.Deep eutectic solvents (DESs) are a potentially high-value lignin extraction methodology. DESs prepared from choline chloride (ChCl) and three hydrogen-bond donors (HBD)—lactic acid (Lac), glycerol, and urea—were evaluated for isolation of willow ( Salix matsudana cv. Zhuliu) is a new willow variety in the Salicaceae Salix spps., with high planting density, fast growth, strong resistance, and high survival rate, that has been widely applied in wind prevention and sand fixation. Moreover, willow has a good potential for the pulp and paper industry because it is fast-growing, particularly in China [Lignocellulosic biomass is comprised of cellulose, hemicelluloses, and lignin. These biopolymers are promising building blocks for the burgeoning bioeconomy ,2,3. Wilin China ,5.Ionic liquid (IL) pretreatment of biomass has attracted broad scientific attention despite high costs and possible toxicity ,7. Howev13C-NMR, and 31P-NMR.To date, there has not been a detailed investigation on applying DESs for extracting lignin from willow. In this work, three different DESs were used, and the effects of treatment time and temperature of the optimal DESs on extraction of lignin from willow was investigated. The lignin extracted from willow by DES treatment that was defined as DES-lignin was characterized by Fourier transform infrared spectroscopy (FT-IR), To investigate the effect of different DES and temperature on the extraction of willow lignin, three DES mixtures with the same mole ratio of choline chloride (ChCl) to three HBDs (molar ratio 1:2): glycerol (ChCl-Gly), urea (ChCl-U), and lactic acid (ChCl-Lac) were prepared. The treatment of willow by each DES was conducted at four different temperatures: 90, 100, 110, and 120 °C over 6 h.The solid residue and DES-Lignin yield are shown in Six different mole ratios of ChCl to Lac  were carried out and compared at 120 °C and 6 h. As a control, pure lactic acid (Lac) treatment on the removal of willow components was also studied. In ChCl-Lac with a molar ratio of 1:10 was among the most effective DESs for willow treatment. The amount of lignin extracted at this latter ratio increased significantly with increasing treatment time from 6 h to 12 h . HoweverThe chemical composition of the solid residue and dissolved carbohydrates after ChCl-Lac (1:10) treatment at 120 °C for 12 h was characterized for sugars and lignin (To investigate the purity of lignin using ChCl-Lac (1:10) treatment at 120 °C for 12 h, the acid insoluble lignin (AIL), acid soluble lignin (ASL), sugars, and ash content in DES-Lignin were measured . Only 0.−1 in the solid residue were significantly reduced, which indicated that most of the lignin was removed from willow after ChCl-Lac (1:10) treatment [The FT-IR spectra of the initial sample, solid residue, and DES-lignin are shown in reatment ,28,29.−1 indicated that the benzene ring skeleton of lignin was left intact. In addition, the higher intensity of the bands at 1325, 1220, and 1120 cm−1 indicated that the syringyl unit played a dominant role. In contrast, the lower intensity of the bands at 1270 and 1035 cm−1, and the absence of the band at 1156 cm−1 suggested that a reduced level of guaiacyl units and a negligible amount of p-hydroxyphenyl units were in the DES-Lignin. The bands at 1426, 1373, 1164, and 1056 cm−1 were absent, suggesting most of the carbohydrates were not present in the DES-Lignin. However, a negligible amount of hemicellulose present in DES-Lignin due to the existence of the band at 1710 cm−1 was consistent with the results of previous lignin purity analyses [The bands at 1600 and 1510 cmanalyses ,31.13C-NMR spectra of DES-Lignin are presented in p-coumaric acid was in DES-Lignin [The S-Lignin .31P-NMR was further used for the quantitation of hydroxyl groups in DES-lignin, and the functional group content from quantitative 31P-NMR is listed in −1) was higher than that of poplar alkali lignin (AL) or other biorefinery lignins [ lignins , suggestSalix matsudana cv. Zhuliu) was harvested and sawed in a forestry center in Dingxi, Gansu Province, China. The representative sample of the sawed willow trunk  was collected and air-dried at ambient temperature in lab for one month. The air-dried sample was cut to 3–5 cm in size and a part of the sample was ground in a star mill. The 40 to 80 mesh fractions were used as the raw material for composition analysis and the subsequent DESs treatment. The chemical composition of willow (wt %) was determined according to the Laboratory Analytical Procedure for Biomass provided by NREL [The five year old raw material of willow ( by NREL  and the Three different DESs were prepared: choline chloride to glycerol (ChCl-Gly) (molar ratio 1:2), choline chloride to urea (ChCl-U) (molar ratio 1:2), and choline chloride to lactic acid (ChCl-Lac) . The solutions were stirred in an oil bath at 60 °C to form a homogeneous liquid without of any solid particles. The mixture was cooled down and stored in a desiccator for further use.g:g). The vessel was then heated by oil bath to the desired temperature to start the reaction. The effect of HBD types , the molar ratio of choline chloride to lactic acid , the reaction temperatures (90–120 °C), and the reaction time (6–42 h) were examined. Each run was conducted in at least triplicate until the data had good reproducibility.Prior to DES treatment, the sample was Soxhlet-extracted with the mixture of benzene and 95% ethanol for 8 h to remove extractives. After that the sample was dried in the vacuum oven at 60 °C for 12 h. For each run, 2.5 g of extractive free sample was added into DESs solvent in reaction vessels with the solvent to solid ratio of 30:1  water (2000 mL) to precipitated lignin. After 24 h, lignin was collected after centrifugation, and washed with a water/ethanol mixture (9:1) . The DESSolid residue yield was calculated as follows:DES-Lignin yield was calculated as follows:The chemical composition of the solid residues and DES-Lignin were analyzed according to the NREL methods . DissolvFor convenient comparison, the content of monosaccharides was calculated as follows:−1.The DES-Lignin was characterized by FT-IR . The dried samples were embedded in KBr pellets at concentrations of ~1 mg/100 mg KBr. All spectra were recorded in absorption band mode over the range of 4000 to 600 cm13C-NMR spectra were recorded on a 400 MHz Bruker AVANCE III NMR spectrometer . Samples of 60 mg were completely dissolved in 500 μL of dimethyl sulfoxide (DMSO-d6). The chemical shifts were calibrated relative to the signals of DMSO-d6, which was used as an internal standard at 39.5 ppm for the 13C-NMR spectra. The acquisition time was 1.4 s and the relaxation time was 1.7 s.The 31P-NMR spectra were recorded on a 400 MHz Bruker AVANCE III NMR spectrometer . Twenty-five milligrams of sample were completely dissolved in 400 μL of anhydrous pyridine and deuterated chloroform . Then 150 μL of cyclohexanol/chromium(III) acetylacetonate solution  was added as an internal standard and relaxation reagent, respectively. The mixture was reacted with 75 μL of phosphating reagent  for about 5 min and then was transferred into a 5 mm NMR tube for subsequent NMR test.The Salix matsudana cv. Zhuliu) at high yield and high purity. Reaction conditions, including reaction time, temperature, and HBD types and dosage, were all shown to be important for extraction. DESs formed by choline chloride and lactic acid (molar ratio = 1:10) at 120 °C and 12 h were appropriate and resulted in 91.82% DES-lignin yield. Extracted DES-lignin displayed high purity (94.46%) with unique structural properties as indicated by FT-IR spectroscopy, 13C-NMR, and 31P-NMR. Moreover, more than 80% of the initial glucan was recovered as a solid residue for further enzymatic hydrolysis or chemical conversion.Deep eutectic solvents (DESs) were shown to be an effective method for the isolation of lignin from willow lignocellulosic biomass (